RcAlb-PepIII bioinspired from the antimicrobial protein Rc-2S-Alb was designed, synthesized, and tested against barber pole worm Haemonchus contortus. The physicochemical properties of the peptide, the 3D structure model, the egg hatch inhibition, and larval development inhibition of H. contortus were carried out. Additionally, the ultrastructure of the nematode after treatment with the peptide was evaluated by atomic force microscopy. The RcAlb-PepIII inhibited the larval development of H. contortus with an EC50 of 90 µM and did not affect egg hatch. Atomic force microscopy reveals the high affinity of RcAlb-PepIII with the cuticle of H. contortus in the L2 stage. It also shows the deposition of RcAlb-PepIII onto the surface of the cuticle, forming a structure similar to a film that reduces the roughness and mean square roughness (Rq) of it. In conclusion, the bioinspired RcAlb-PepIII has the potential to be used as a new anthelmintic compound to control gastrointestinal nematode parasites.Resistant nematodes are not affected by the most common drugs commercially available. In the search for new anthelmintics, peptides have been investigated. Here, a linear synthetic peptide named  H. contortus is a hematophagous gastrointestinal nematode from a ruminant abomasum with the highest impact on livestock production. Several studies have reported multi-drug H. contortus resistance on strains of small ruminants’ farms from all continents worldwide [Infection caused by multi-drug resistant nematodes in ruminants led to a negative impact on livestock production and significant economic losses worldwide ,2 . They are part of the first line of defense toward microbes such as bacteria, fungi, and protozoa or viruses [Antimicrobial peptides are natural antibiotics produced by all living organisms, including plants, which are known to contain more than 273 compounds   revealed insights about the mechanism behind RcAlb-PepIII against the gastrointestinal nematode H. contortus, revealing the biotechnological potential of RcAlb-PepIII to develop peptide-based anthelmintic drugs. Our research group purified a potent antimicrobial protein from c-2S-Alb , which wopicalis , but notRcAlb-pepIII was designed as previously described [Rc-2S-Alb [The peptide escribed  based onc-2S-Alb .http://www.imed.med.ucm.es/Tools/antigenic.pl, accessed on 15 January 2021); Peptide Cutter tool ; and HLP  [RcAlb-PepIII was chemically synthesized by GenOne , which analyzed their quality and purity (≥95%) by reverse-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry. The three-dimensional (3D) structures were produced using the PEPFold server [Predicting Antigenic Peptides (ry 2021) . These td server .RcAlb-pepIII peptide was solubilized in the 50 mM PBS 7.4 pH buffer with a concentration ranging from 50 to 300 µM. Third-stage larvae (L3) of a monospecific strain of H. contortus  were used to experimentally infect two six-month-old lambs (Santa Inês breed), which were maintained in a metabolic cage. The infected lambs received hay and water ad libitum and 1% of its live weight of commercial feed with 20% crude protein. H. contortus eggs were obtained according to Coles et al. [To this assay, the s et al. . Experimg) for 3 min; floating eggs were recovered using a 25 µm sieve. Eggs were washed three times to eliminate the remaining salt and re-suspended in distilled water. A serial dilution in PBS pH 7.4 was performed, with a final concentration of peptides ranging from 50 to 300 µM. In the egg hatch test (EHT), approximately 100 eggs per well were plated in 96-well flat-bottom plates, with four replicates, and the different dilutions were added. The plate was incubated at 27 °C and Relative Humidity (RH) >80%; after 48 h, the eggs and larvae were quantified under an inverted microscope [Freshly collected feces were mixed in warm water (37 °C), and the eggs were recovered from the solution in 25-µm sieves. Afterwards, eggs were added to a saturated NaCl solution and centrifuged (3000× croscope .H. contortus eggs were obtained as previously described. To 96-well flat-bottom plates were added 100 eggs/well (100 µL), which were incubated at 27 °C for 24 h until the eggs reached the first-stage larvae (L1). Then, 40 µL of Escherichia coli medium (autoclaved Escherichia coli) at 0.15 mg mL−1, yeast extract at 8 mg mL−1 in saline 0.9%, Earle’s solution at 0.22 mg mL−1, and amphotericin B (Sigma A2942) at 0.018 mg mL−1 were added in each well. The peptides were diluted in PBS pH 7.4 (as discussed above), and 110 µL were added to reach six final concentrations among 50 to 300 µM. Four replicates of each concentration were performed. The 96-well plates were incubated at 27 °C for six days, and then, L1 and L3 were counted under an inverted microscope [croscope .RcAlb-PepIII and assessment of the topography and mechanical properties of larvae with and without treatment. The larval development assay was carried out as previously described using the peptide in the IC50 concentration. Phosphate buffer solution (pH 7.4) was used as control. Eight replicates of the experiment were carried out. After 48 h, the control was observed under an inverted microscope to confirm the second-stage larvae’s presence (L2). The content of four replicates and its control were collected and fixed in 10% paraformaldehyde (1:1). Then, 120 h after starting the test, the other wells containing L3 were fixed as described.These AFM measurements were performed for the characterization of H. contortus control and treated were deposited in 13 mm glass coverslips previously treated with poly-l-lysine 1% , aiming to improve the substrate’s adhesion and avoid the samples being dragged during scanning. AFM measurements were performed using a Multimode 8 microscope  in PeakForce Tapping Quantitative Nanomechanics mode, in air (23 °C temperature and 44% humidity), using silicon probes with a nominal spring constant of 0.4 N/m and nominal tip ratio of approximately 2 nm. However, the actual spring constant of each probe used in this work was measured by the thermal noise method. Images were taken at approximately the center region of the larvae, with a size of 1 µm × 1 µm for better visualization of peptides on larvae cuticles. The resolution of images was 256 × 256 force curves [The samples of L2 and L3 e curves . The roughness values were calculated from each pixel in the entire AFM height maps, using NanoScope Analysis Software 2.0 (Bruker) according to Equation (1):z is each pixel height value and N is the total number of pixels . To avoid changes in the real values, no image pretreatment was performed on the maps before the calculation of this parameter.That defines the root mean square average of height deviations taken from the mean image data plane, where The adhesion and stiffness data of the samples were calculated from each of the force curves obtained on all samples under study. For the calculation of the adhesion between the probe and the sample surface, the most negative value of force (cantilever deflection) obtained from the retraction curve was considered. These values represent the resistance of the AFM probe to leave the sample surface. For the calculation of the surface stiffness, the slope values in the contact portion of the approach curves were considered, in intervals of 30–70% of deflection after the contact point of the tip with the sample.RcAlb-PepIII (1 mg mL−1) was deposited in previously cleaved mica and analyzed in the AFM under the same conditions previously described. The solution was left in the mica until completely dry and then analyzed on the AFM. For the peptide characterization, 10 μL of To evaluate the hemolytic potential, the peptides  were incubated with rabbit red blood cells for 1 h at 37 °C . ExperimRcAlb-PepIII was assessed by cell viability assay using 3--2,5-diphenyltetrazolium bromide  described by Souza et al. [The toxicity of a et al. . Cell viRcAlb-PepIII was designed from the primary structure of Rc-2S-Alb [RcAlb-PepIII was described as an anthelmintic peptide. Based on that, some of the physicochemical and biological properties of RcAlb-PepIII were compared with Kalata b1, which is a well-known anthelmintic peptide [RcAlb-PepIII is smaller and basic  compared to Kalata b1 . RcAlb-PepIII has a higher hydrophobic potential and is positively charged, whereas Kalata b1 is negatively charged . It is clear and expected that Kalata b1 has a larger structure than RcAlb-PepIII  due to the RcAlb-PepIII peptide effect. treated C,D and u treated A,C with reatment A, the direatment C, suggesreatment C points  samples A, the me samples C, the RqRcAlb-PepIII on H. contortus L3 was also verified  and decreasing the adhesion in the L3 stage (control: 0.8 ± 0.23 nN and treated: 0.62 ± 0.3 nN) A.For stiffness results B, in theRcAlb-PepIII, bioinformatics analyses revealed no toxicity potential, which was experimentally proven. None of the tested concentrations  higher than IC50, the peptide has no toxicity of any kind to mammalian cells. Regarding the toxicity to Vero line cells, the concentrations of 125, 250, and 500 µM were not toxic  called cycloviolacin are stronger than Kalata peptides. For instance, cycloviolacin O2, O3, and O8 have IC50 values against H. contortus of 0.12, 0.21, and 0.24 µM [The lower activity of y et al.  presentes et al. , RcAlb-PRcAlb-PepIII films-like structure. AFM analysis in this work was also an efficient tool to verify the affinity of RcAlb-PepIII with the cuticle of H. contortus in different development stages. AFM analyses revealed changes in the nematode’s cuticle ultrastructure and surface roughness of the L2 stage of H. contortus. Those film-like structures or surface wear processes reduce the Rq values  and in the surface roughness show the evident structural changes promoted by the treatment. The sheath permeability can be higher in the L3 stage when compared to the L2, which would promote a rapid absorption of the peptides by the surface and make it difficult to observe the film on it. It is necessary to consider that this hypothesis is reinforced by the fact that the same treatment time is employed in both stages. These ultrastructural changes suggest wear of the nematode surface, in both stages of development, corroborating the findings of Svangard et al. , which sRcAlb-PepIII treatment promoted different physical changes, increasing the adhesion in the L2 stage and decreasing the adhesion in the L3 stage. It is important to notice that adhesion forces are a combination of electrostatic, van der Waals, capillary, and escape forces promoted by the chemical bonds [RcAlb-PepIII treated samples can means changes in the physical properties of the sample cuticle, indicating the presence of the peptide on the cuticle and/or physical properties changes of its structures due to wear.It was observed that for each stage of development, the al bonds . EspeciaRcAlb-PepIII treatment, the cuticle’s stiffness values increase. This result is promising because at this development stage, the nematode has a sheath, which must be removed to promote its infection stage (L3 unsheathed) [For stiffness results, in the L2 stage, the scatter plots suggest greater uniformity of the stiffness values after treatment. However, in the L3 stage, after heathed) . A greatRcAlb-PepIII and Kalata B1, the mechanism of anthelmintic activity is entirely different. It must be noticed as an exciting point in the mode of action of RcAlb-PepIII. The AFM analysis of RcAlb-PepIII showed that it organizes itself in a hexagonal shape forming circular structures similar to honeycombs (RcAlb-PepIII (RcAlb-PepIII were covered with a film-like structure that has the same hexagonal shape presented by RcAlb-PepIII alone. Apart from the physicochemical and structural similarities shared by b-PepIII B,D. The RcAlb-PepIII. However, they all share a problem, cytotoxicity, with human cells [Indeed, the cyclic peptides presented here are stronger than an cells . Kalata an cells . For exaan cells . Regardian cells . Kalata an cells . DespiteRcAlb-PepIII has an advantage compared to cyclic peptides. Despite having a higher IC50 than the cyclotides above cited, RcAlb-PepIII presented no toxicity to mammalian erythrocytes and Vero line cells at concentration four times higher than IC50 RcAlb-PepIII (H. contortus in small ruminants. As it stands, RcAlb-PepIII has an IC50 of 90 µM against H. contortus (In that matter, b-PepIII , suggestontortus . H. contortus. AFM analyses of the peptide alone and in contact with the cuticle of L2 provided new insights about the anthelmintic activity mechanism displayed by a peptide, which was not described before. The utmost importance is that RcAlb-PepIII did not present any toxic effect on mammalian cells, even at a concentration of five times the IC50. Altogether, the results presented here come up with the biotechnological potential of RcAlb-PepIII in control of H. contortus without any collateral effects to the hosts.Here, we showed for the first time the anthelmintic activity of a synthetic linear peptide against


Intrastriatal embryonic ventral mesencephalon grafts have been shown to integrate, survive, and reinnervate the host striatum in clinical settings and in animal models of Parkinson’s disease. However, this ectopic location does not restore the physiological loops of the nigrostriatal pathway and promotes only moderate behavioral benefits. Here, we performed a direct comparison of the potential benefits of intranigral versus intrastriatal grafts in animal models of Parkinson’s disease. We report that intranigral grafts promoted better survival of dopaminergic neurons and that only intranigral grafts induced recovery of fine motor skills and normalized cortico-striatal responses. The increase in the number of toxic activated glial cells in host tissue surrounding the intrastriatal graft, as well as within the graft, may be one of the causes of the increased cell death observed in the intrastriatal graft. Homotopic localization of the graft and the subsequent physiological cell rewiring of the basal ganglia may be a key factor in successful and beneficial cell transplantation procedures. Parkinson’s disease (PD) is the most common progressive neurodegenerative movement disorder, characterized mainly, but not exclusively, by a marked ongoing degeneration of mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc), leading to a loss of dopamine axon terminals and a reduction in dopamine in the target structure, the striatum . ClinicaIn this study, we compared side-by-side the anatomical and functional effects of fetal VM grafts placed in the striatum or the SNpc of 6-OHDA-lesioned mice. The functional effects were tested using a battery of simple to complex behavioral tests and electrophysiological measurements, while the neuroanatomical integration of transplanted cells was evaluated by immunohistochemical analysis.n = 3 intrastriatal; n = 3 intranigral) and 56 with tyrosine hydroxylase-GFP (TH-GFP) mouse fetal VM tissue . The 6 mice transplanted with VM fetal tissue from beta-actin-GFP mice were used for immunohistological analysis of the transplant. A total of 39 mice were used for behavioral tests . A total of 31 mice were used for amphetamine-induced rotation to evaluate the effects of removal of the intranigral graft . In total, 37 mice were used for the electrophysiological studies . An amount of 1 mouse from each transplanted group  was used for electron microscopy.Animal experimentation and housing were carried out in accordance with the guidelines of the French Agriculture and Forestry Ministry (decree 87849) and the European Communities Council Directive (2010/63/EU). The procedures referenced under the file number APAFIS#4928-20 16041 117503028 v3 were approved by ethics committee N°84 COMETHEA Poitou-Charentes. All mice were housed under a standard 12 h light: dark cycle. The animals had ad libitum access to food and water. All efforts were made to reduce the number of animals used and their suffering. A total of 118 animals were used in this study . During 6-OHDA Lesioning: Adult female C57BL/6 mice  weighing 20–25 g at the time of surgery were used in this study. Under ketamine-xylazine anesthesia , mice received into the left SNpc 1 μL 6-OHDA  at the following coordinates (in mm relative to bregma): AP, −3.1; ML, 1.4; DV, 3.8. The drug was injected slowly (0.5 µL/min), and the cannula was left in place for 5 min to allow diffusion of the solution before being retracted.Transplantation: To facilitate the identification of the transplanted dopamine neurons and their axonal projections within the host brain, donor VM tissue cells were isolated from transgenic mice overexpressing GFP under the influence of the TH promoter [promoter . To analpromoter . Transplpromoter ,48. TimeCylinder: Forelimb use asymmetry during vertical exploration was assessed using the cylinder test . The mic2) of corresponding width was placed on the beam surface to increase the difficulty in such a way that, in a forward motion of the forelimb, the paw could be “placed” on the grid or “slipped” through the grid. Two days before testing, the animals were trained without the grid with two assisted trials and were able to traverse the beam until they completed five unassisted runs across the entire length of the beam. On the second day of training, the animals were allowed to run five trials. Prior to starting the test, the mice were allowed to accomplish two trials on the beam and two trials with the grid. The mice were then videotaped while traversing the grid-surfaced beam for a total of three trials. The videotapes were viewed and rated in slow motion for measuring time to traverse, and for each forelimb, the percentage of foot slips was calculated according to the following formula: % of foot slips  = number of foot slips / + number of foot slips ) × 100.Challenging beam traversal: Motor performance was measured 6 weeks post-transplantation using a beam-walking test ,56 with Apomorphine- and amphetamine- induced rotations: Apomorphine- and amphetamine -induced rotational behaviors were assessed using an automatic rotameter  according to Ungerstedt and Arbuthnott (1970) . Full boStaircase: The staircase test was designed for the objective assessment of independent forelimb use in skilled reaching and grasping abilities ,60. The Videotaping and kinematics: Video recordings were conducted with a miniature monochrome camera  positioned on the right side of the apparatus. The camera was set to 25 frames/s with a resolution of 500 × 582. Each right grasping movement was divided frame-by-frame, captured, and saved using an ethological keyboard . A movement component rating scale derived from Eshkol–Wachman Movement Notation (EWMN) allowed us to decompose and divide the entire grasping movement into three task phases: the reaching movement ranging from the moment the paw was lifted from the ground until it touched the pellet; the grasping movement corresponding to the closing of the digits; and the retrieval movement up to the release of the pellet into the mouth. To measure the time required for each task phase, we selected right successful reaches (the mouse retrieved the pellet directly to its mouth) on the third step of the right staircase (the step most frequently used). Data were collected the eighth week post-transplantation. In the event that no successful grasping was found, we used the data from the seventh week post-transplantation. One control, two lesioned, two intrastriatal-, and four intranigral-grafted mice were excluded from the analysis because they did not exhibit any successful grasping on the third step of the staircase. The time required to complete each task phase was calculated according to the following formula: (number of frames constituting the phase − 1) × 40 ms. Analysis of the length of the reaching movement trajectory was performed after importing corresponding frames into ImageJ 1.48V software . The pellet position on the third step of the staircase was used as a reference point then the tip of the third finger was pointed on the successive frame, and the x/y coordinates were determined, allowing us to draw Cartesian trajectories of each reaching movement ,62.In order to test the effects of a single injection of L-DOPA on motor-skilled reaching and grasping abilities, the staircase test was performed on two consecutive days (16 weeks post-grafting). L-DOPA methyl ester  and the peripheral DOPA decarboxylase inhibitor benserazide-HCl  were freshly dissolved in 0.9% saline immediately prior to use. On the first day, the mice were tested on the staircase for 10 min in order to measure motor performances 16 weeks after transplantation. On the second day, the mice were injected with drugs  30 min prior to placing them on the staircase for a 10 min session in order to cover the period of maximal drug effect. During the analysis of the skilled reaching abilities, one lesioned and two intranigral-grafted mice were excluded from the count because they did not demonstrate entry into the corridor of the device.Before surgery, the mice were deeply anesthetized with urethane (1.8 g/kg) injected i.p. before being secured to a stereotaxic frame and maintained at 37–38 °C with a heating pad. A mouse brain stereotaxic atlas  was usedElectrophysiological single-unit activity was recorded in the dorsolateral striatum (DLS) using electrodes pulled from borosilicate glass capillaries  with a P-97 Flaming Brown micropipette puller . The tip of the electrode was broken to a diameter of 2 µm, and the electrode was filled with 0.4 M NaCl solution containing 2.5% neurobiotin . Electrodes had an in vivo resistance of 12–20 MΩ. Recording electrodes were lowered in the DLS ipsilateral to the lesion or transplantation site  at a depth comprised between 2 mm and 3 mm from the brain surface.Neuronal activity was amplified 10 times, filtered (bandwith: 300 Hz–10 kHz), and further amplified 100 times . The signal was digitized  and acquired on a computer using Spike2 software. Recorded neurons were juxtacellularly labeled with neurobiotin (Vector Laboratories), as described elsewhere . BrieflyOrthodromic stimulations of the cortex ipsilateral to the recording site were performed using a concentric bipolar electrode  implanted in the motor cortex in order to evaluate the response of striatal neurons to cortical input. Stimulations (duration: 0.5 ms) were applied every 3 s using an external stimulator  triggered by a 1401 Plus system (Cambridge Electronic Design Ltd.), as previously reported ,66,67.The recordings were analyzed offline using Spike2 7.0 (Cambridge Electronic Design Ltd.). The action potential duration was measured from the positive to the negative pic. In order to assess neuronal activity, the action potential mean firing rate was measured for 1 min periods. The stimulation threshold was determined by measuring the minimal stimulation intensity required to evoke a spike response with a probability close to 50%.Tissue processing: The mice were anesthetized and transcardially perfused with 150 mL saline (0.9%) followed by 200 mL ice-cold PFA (4%). The brains were removed and postfixed overnight in 4% PFA and then cryoprotected overnight in sucrose (30% in 0.1 M PB). The brains were cut into 6 series at a thickness of 40 μm on a freezing microtome  and stored in a cryoprotective solution .Immunohistochemistry: Free-floating sections were washed in PBS  and incubated for 1 h at room temperature (RT) in a blocking solution containing 5% BSA and 0.3% Triton X-100 in PBS 0.1 M at pH 7.6. Primary antibodies diluted in blocking solution were applied for 2 h at RT and overnight at 4 °C. After three washings in PBS, appropriate secondary antibodies coupled to Alexa Fluor fluorochromes  were diluted in PBS and applied for 1 h at RT. The sections were rinsed and mounted on gelatinized slides before being coverslipped with DePeX (Sigma-Aldrich) mounting medium. The following antibodies were used in this study: goat anti-GFP , rabbit anti-GFP , chicken anti-GFP , mouse anti-TH , rabbit anti-Girk2 , rabbit anti-calbindin , rabbit anti-DAT , mouse anti-NeuN , chicken anti-GFAP , and rabbit anti-Iba1  Rabbit anti-CD86  and goat anti-Arg1  were used for neurotoxic and neuroprotective phenotypes, respectively. Rat anti-C3  and rabbit anti-CD109  were used for A1 and A2 phenotypes, respectively.Electron microscopy: The mice were perfused transcardially with 150 mL of saline (0.9%) followed by 200 mL fixative . The brains were postfixed overnight in 2% PFA and cut into 60 µm coronal sections with a vibrating microtome . The sections were preincubated for 90 min in a blocking solution  in 0.1 M PBS; pH 7.4). For immunogold detection of GFP, the vibratome sections were incubated overnight at 4 °C with rabbit anti-GFP , rinsed in PBS, and incubated for 4 h at RT in goat anti-rabbit IgG conjugated to ultra-small gold particles . The immunogold signal was intensified using a silver enhancement kit . After several washes, the sections were postfixed in 2% osmium tetroxide and dehydrated in an ascending series of dilutions of ethanol, including 70% ethanol containing 1% uranyl acetate and propylene oxide. Then, the sections were impregnated in LR White resin overnight (Sigma-Aldrich), mounted on glass slides, and cured at 60 °C for 24 h. Areas of interest were first visualized in a light microscope and then cut out from the slide and glued to blank cylinders of resin. Serial ultrathin (60 nm) immunostained sections were cut with a UC6 Ultramicrotome (Leica Biosystems), contrasted with lead citrate, and observed with an electron microscope n = 9). To estimate the total number of dopaminergic neurons found in the transplants, an extrapolation of the total number of GFP+ cells contained in one series of the sections from intrastriatal (n = 10) or intranigral (n = 10) grafts immunohistochemically labeled for GFP was sampled using confocal microscopy. The method of Abercrombie (1946) [n = 7) or intranigral (n = 6) grafts immunohistochemically labeled for GFP and Girk2 was sampled using confocal microscopy, and co-localization was confirmed by z-axis analysis. The same procedure was used to assess the number of GFP+ cells that co-expressed calbindin or dopamine transporter (DAT) in grafts immunohistochemically labeled for these markers. GFP+ fibers were quantified for each animal in five sections for each area using a high-magnification objective (20×).Immunofluorescence-treated sections were examined with an Axio Imager M2 Apotome microscope . The selected sections were further analyzed and photographed with an FV1000 confocal laser-scanning microscope . The extent of dopaminergic depletion was analyzed by quantifying the number of TH+ neurons remaining in injured SNpcs compared to intact SNpcs in a 1:6 series of sections (e (1946)  was usedAll data were expressed as mean ± the standard error of the mean (SEM). Normality was checked using the Shapiro–Wilk test. The Mann–Whitney U test was used to compare the average number of GFP+ and NeuN+ cells in the two groups of grafted animals . For the statistical significance of glial fibrillary acidic protein (GFAP)+ and ionized calcium binding adaptor molecule 1 (Iba1) + cells in the host, transplant, and two grafted groups, the Holm–Sidak method was used with alpha = 0.05. For behavioral studies, the following statistical tests were performed: (i) a Kruskal–Wallis test to compare the time required to perform movements and the staircase test with or without L-DOPA injections; (ii) a one-way analysis of variance (ANOVA) with group as a factor for the challenging beam test, the staircase test, and analysis of the length of the reaching-movement trajectory 8 weeks post-transplantation; and (iii) repeated measures of ANOVA using group  or time (rotation tests) as factors. Post hoc analysis was conducted with Dunn’s model for nonparametric tests and Fisher’s protected least significance difference (PLSD) test or the Dunnett test, as appropriate.p < 0.05.For electrophysiological experiments, statistical analyses were performed using a one-way ANOVA followed by a Tukey post-hoc test. The statistical significance level was set at n = 9), the number of TH+ neurons in lesioned SNpcs was 114 ± 41 compared to 1259 ± 29 in the intact side (91.4 ± 3% decrease), which is in accordance with our previous findings [n = 21) and intrastriatal (n = 22) transplants. Intranigral grafts were mainly located in the dorsal part of the SNpc, and in some cases, a few GFP+ cells were also found along the injection tract. Intrastriatal grafts were located in the dorsal or medial parts of the striatum near the lateral ventricle. Some isolated GFP+ cells were observed at the host–graft interface. Intrastriatal grafts (n = 10) contained significantly less (−48.72%) GFP+ neurons (732 ± 231) than intranigral ones  . Almost all GFP+ cells within the intranigral or intrastriatal transplants co-expressed TH (>95%), which is in agreement with previous studies [Four months after 6-OHDA injection  and intranigral (n = 6) grafts  grafts c,h; thes) grafts d,e, a fe) grafts i,j, were) grafts b,g, and p = 0.346 and p = 0.137, respectively; Mann-Whitney test) in both transplantation conditions.More than half of the GFP+ dopaminergic neurons were positive for Girk2 in both transplantation conditions. Indeed, with the intrastriatal transplants, we found that among 653 ± 156 GFP+ neurons, 345 ± 85 neurons were also positive for Girk2, and among 342 ± 74 of GFP+ neurons, 154 ± 37 neurons were positive for calbindin k,l. WithThe lower number of TH-GFP+ cells in intrastriatal grafts led us to delve further into the cellular composition of the grafts. In order to identify all transplanted cells after grafting, VM donor tissues were isolated from transgenic mice over-expressing GFP under the influence of beta-actin promoter.n = 6) and intrastriatal (n = 6) grafts. We reported a significant increase in the number of NeuN+ cells in the intranigral grafts (1490 ± 194) compared to the intrastriatal grafts   .n = 6, * p < 0.01). We also observed a significant increase in the number of GFAP+ cells in the intrastriatal grafts (622 ± 124) compared to the intranigral grafts    compared to the intranigral grafts  h–m. In o< 0.006) n, indican = 6, * p < 0.05)   compared to the intranigral grafts  o. There < 0.006) o. We furp < 0.006). We also observed a higher number of Iba1+ cells co-expressing Arg1 in intranigral grafts .As the level of inflammation within the graft was different between the intrastriatal and intranigral transplants, we investigated the pro-inflammatory vs. anti-inflammatory profiles of activated microglia and astrocytes in the grafts. Microglial cells respond to brain injury by becoming activated and shifting between neurotoxic (detected with CD86) or neuroprotective phenotypes (detected with arginase-1). We observed that the percentage of Iba1+ cells co-expressing CD86 was higher in intrastriatal grafts , and the percentage of GFAP+ cells co-expressing the C3 marker was higher in intrastriatal grafts . The percentage of GFAP+ cells co-expressing the CD109 marker was very low and not significantly different between the two graft conditions ), which induce the rapid death of neurons and oligodendrocytes, and neuroprotective A2 astrocytes (detected with CD109), which promote neuronal survival and tissue repair ,77. We o44 ± 6%, a–d,a’–d’GFP fibers from the intrastriatal a–c or inIn order to investigate to which level transplanted dopamine neurons integrated into the host circuits, we assessed the reciprocal synaptic contacts between intrastriatal or intranigral transplants and the host by electron microscopy four months post-grafting , Table 21,28 = 5.82; p < 0.05). Lesioned animals (n = 9) did not show any spontaneous improvement in their rotation scores between the first testing session (61 ± 15) and the second one . On the other hand, the intranigral-transplanted mice (n = 11) net contralateral turns were significantly reduced by 55% (20 ± 12) in comparison to their pre-grafting scores . In the same way, the intrastriatal-transplanted mice (n = 11) rotations were also significantly reduced by a similar proportion of 68% than before grafting . In another set of mice, we evaluated amphetamine-induced rotations both before and after transplantation . Lesioned animals (n = 5) did not show any significant difference in their rotation scores between the first testing session (346 ± 49) and the second one . On the other hand, the intranigral-transplanted mice (n = 5) turns were significantly reduced (24.8 ± 14) in comparison to their pre-grafting scores . In the same way, the intrastriatal-transplanted mice (n = 5) rotations were also significantly reduced in comparison to their pre-grafting scores .The net contralateral rotation response to apomorphine during a 60 min session was recorded pre-grafting and thirteen weeks after transplantation in the same animals, as shown in antation b during n = 8; lesioned, n = 7; intrastriatal-grafted, n = 11; intranigral-grafted, n = 10) in the use of the forepaws contralateral to the lesion site  and a significant interaction between the groups and times of testing  . Ten weeks after transplantation, both lesioned and intrastriatal-grafted mice showed a marked impairment of the contralateral forelimb contacts in comparison to control mice . However, no difference was observed between intranigral-grafted and control mice, indicating a recovery in fine motor skills was required for this motor task.At three and ten weeks post-grafting, the mice were tested for forelimb asymmetries using the cylinder test. We found a difference between the groups  c. At thr3,35 = 3.45, p < 0.05). Indeed, 6-OHDA-lesioned mice needed a much longer time to cross the beam than intranigral-grafted  and control  mice. No significant difference was found between the control group and either the intrastriatal (p = 0.1024) or intranigral (p = 0.4079) group , one-way ANOVA revealed a main effect between groups in the percentage of foot slips made with forelimbs that were contralateral to the lesion. . Post-hoc analysis indicated that lesioned mice exhibited a significantly greater percentage of contralateral forelimb errors in comparison to the control group (p < 0.05). Intrastriatal-transplanted mice displayed significantly more contralateral forelimb errors than the control group (p < 0.01) and were not different from lesioned mice . Intranigral-grafted mice displayed a significant decrease in contralateral forelimb errors in comparison both to the lesioned and intrastriatal-transplanted groups (both p < 0.05) and were not different from control mice , indicating a restoration of contralateral forelimb use following intranigral transplantation.Six weeks after transplantation, we observed a significant group difference in the challenging beam test in the time required to cross the beam  group . In orden = 8; lesioned, n = 9; intrastriatal-grafted, n = 11; intranigral-grafted, n = 11). Prior to surgery, the animals were trained on the staircase apparatus in order to assess baseline performance and were thereafter tested once a week during six consecutive sessions (from weeks three to eight post-transplantation) divided into two blocks of three sessions (from weeks three to five and from weeks six to eight). No significant difference between groups was found in the baseline performance , indicating that all groups acquired the test similarly . Six to eight weeks post-transplantation, one-way ANOVA revealed difference between the groups  compared to the control group. However, intranigral-grafted mice showed a significant improvement in grasping performance compared to lesioned and intrastriatal-transplanted mice , and no difference was found between control and intranigral-grafted mice (p = 0.47).Lesions of the nigro–striatal pathway produced skilled-forelimb reaching deficits that we assessed here in four groups of animals . In particular, lesioned mice (n = 7) needed more time than both control (n = 7) and intranigral-grafted (n = 7) mice  to contact the pellet. In addition, this reaching phase was also significantly longer with the intrastriatal-grafted mice (n = 9) in comparison to both control and intranigral-grafted mice . In contrast to the reaching time, the time taken to execute the grasping, the retrieval, or the complete movement was similar between the groups . In conclusion, we found that the time required to achieve the reaching phase was similar between control and intranigral-grafted mice, indicating a marked recovery of movement in this task.We evaluated the kinetics of forelimb movements contralateral to the lesion site from seven to eight weeks post-transplantation. For this, the complete movement was divided into three tasks , and we measured the time required to complete each task phase on the third step of the staircase . We found that the time required to achieve the reaching phase was different between the groups , with the lesioned mice displaying a significant increase in the length of the trajectories compared to both control and intranigral-transplanted mice . Following the same pattern, the trajectories of intrastriatal-grafted mice were also significantly longer than in both control and intranigral-transplanted mice . Interestingly, no difference was found in the length of trajectories between both control and intranigral (p = 0.52) mice or between lesioned and intrastriatal-grafted (p = 0.69) mice. This indicated that only intranigral grafts were able to restore this motor behavior.In order to refine this initial reaching phase, we analyzed the reaching trajectories of each group f. Interen = 8; lesioned, n = 8; intrastriatal-transplanted, n = 11 and intranigral-transplanted, n = 9, p < 0.05, Kruskal–Wallis test). We found a robust 6-OHDA lesion effect in the contralateral forelimb bias between lesioned and control mice (p < 0.05). Additionally, only intranigral-grafted mice displayed significant improvement of their contralateral forelimb bias in comparison to lesioned mice (p < 0.01), and no significant difference was found between control and intranigral-transplanted mice (p = 0.4947). Interestingly, a single injection of L-DOPA improved the performance of the reaching task with impaired limbs of intrastriatal-transplanted mice. The contralateral bias of lesioned and intrastriatal-grafted mice increased from 17.5 ± 6.66% to 34.37 ± 8.09% and from 34.54 ± 12.89% to 43.18 ± 9.59%, respectively, following the L-DOPA injection (p < 0.05), no difference was found among all the other groups.We then analyzed the length of reaching-phase trajectories following a single i.p. L-DOPA injection . For thin = 7) and intrastriatal-grafted (n = 7) mice in comparison to control (n = 7) and intranigral-grafted (n = 7) mice . Without an L-DOPA injection, both lesioned and intrastriatal-grafted mice displayed a significantly longer length of trajectory in comparison to control  and intranigral-grafted mice . However, following a single L-DOPA injection, no difference was found between intrastriatal-grafted mice and control or intranigral-grafted mice . In contrast, the length of trajectory for the lesioned mice was not affected by the treatment and was significantly longer than control, intrastriatal-, and intranigral-transplanted mice . This set of experiments indicated that only intranigral grafts restored normal behavior in this task, and this was not further potentiated by an L-DOPA injection. Intrastriatal grafts were deficient but were able to demonstrate this behavior restored to normal levels by a single L-DOPA treatment.We next conducted a detailed analysis of the grasping movement for each group during the two sessions (without and with an L-DOPA injection) in the staircase test. Sixteen weeks post-grafting and without an L-DOPA injection, we found a similar pattern in the grasping trajectories as described above with very irregular reaching movements for both lesioned ( 7) mice b. Conver 7) mice c. RepeatIn order to confirm that the functional recovery was induced by intranigral grafts, we proceeded to remove the intranigral-grafted transplant by an injection of 6-OHDA 2 months after transplantation. The functional impact of the graft lesion was assessed 4 weeks after 6-OHDA injection using amphetamine-induced rotation. In transplanted-then-lesioned animals, the original motor asymmetry deficit re-appeared, and the number of rotations was no longer different from the performance observed in the pre-graft rotation scores d. Histoln = 14, n = 8, n = 8, and n = 8 mice, respectively).Dopaminergic denervation resulted in an alteration of striatal neuron basal-firing properties, as well as in cortico-striatal information processing ,79. In op = 0.0041) in the lesioned group (0.86 ± 1.47) compared to the control (0.03 ± 0.11) and both grafted groups   c,d. Thisp = 0.0006) in the lesioned and striatal-grafted groups  compared to control and SNpc-grafted animals   e. These  ± 0.23) .The purpose of the present study was to compare the effects of intranigral versus intrastriatal transplantation of fetal VM tissue in a 6-OHDA mouse model of PD. We analyzed graft survival, integration, expression of dopaminergic markers, and projections, as well as behavioral and electrophysiological activity. Our data showed that: (1) fetal VM tissue transplanted either into the SNpc or the striatum is able to survive, re-innervate the denervated striatum, express dopaminergic markers in same proportions, and establish reciprocal synaptic contacts with host neurons. (2) Both graft locations significantly reduced apomorphine- and amphetamine-induced rotations, ameliorated motor performance of simple behavioral tasks, and (3) induced the recovery of striatal firing properties of MSN. In contrast, only intranigral transplantation (4) dramatically increased the number of dopaminergic neurons found within the transplant, (5) sent appropriate projections to the striatum through the mfb, (6) improved recovery of forelimb fine motor skills for complex behavior tasks without further need for L-DOPA treatment, and (7) efficiently normalized cortico-striatal responses.Homotopic placement of the intranigral transplant may expose the cells to appropriate nigral trophic factors and afferent inputs, allowing the better survival of the grafted neurons, as previously indicated ,80,81,82We observed that the axon terminals of both intranigral- and intrastriatal-grafted dopamine neurons formed synapses on dendritic spines and shafts of striatal neurons. These results are in agreement with findings reported in intact animals ,84,85. IThe distribution pattern of dopaminergic terminals in contact with different parts of the striatal neurons is known to impact synaptic functions. Indeed, most dopaminergic inputs that occur on the necks of dendritic spines seem to be more selective, as they prevent excitatory glutamatergic input to the same spines from reaching the dendritic shaft ,86. In cThe anatomical origin of host inputs to grafted neurons is largely influenced by graft placement, as recently shown by Alder et al., 2020 . In theiFetal VM tissue used for transplantation contains both nigral  and VTA  dopamine subtypes. We found that a majority of the dopaminergic neurons grafted both in the striatum and the SNpc expressed the nigral subtype Girk2 in a similar proportion between the two graft conditions. Both in intrastriatal and intranigral transplants, the majority of dopamine neurons that co-expressed Girk2 were found in the periphery of the graft, while dopamine neurons co-expressing the VTA marker calbindin were primarily located within the center of the graft, as previously reported ,69,91.Despite the identical number of cells transplanted in the striatum and in the SNpc, we reported a significant increase in the number of NeuN+ cells in the intranigral grafts compared to the intrastriatal grafts. These results suggest a higher rate of cell survival or a lower rate of cell death of neurons in the intranigral position compared to the intrastriatal position and, thus, a better outcome for the homotopic placement of the graft.Neuroinflammation in PD contributes to disease pathogenesis ,93,94. M.Different activation statuses of microglial cells can promote either neurotoxicity or neuroprotection ,99,100. In addition to microglia, astrocytes also participate in the neuropathology of PD ,102, wheTo evaluate the ability of the two graft strategies to restore physiological striatal activity, electrophysiological investigation of spontaneous striatal activity and cortico-striatal synaptic efficacy were investigated. Our data demonstrated that lesioned animals exhibited increased spike-firing that could be restored to control levels by both striatal and nigral grafts. However, the drop in cortico-striatal transmission efficiency following lesioning was restored only by the striatal graft. Thus, although spontaneous striatal activity was rescued both by striatal and nigral grafts, fine modulation by cortical input was restored only by nigral grafts.In order to evaluate the integration of the grafted neurons into the host motor circuitries, we investigated the ability of the grafts to restore motor function by using a battery of behavioral tests. For all tests used, lesioning of the nigrostriatal pathway induced strong behavioral deficits. Moreover, both the intrastriatal and intranigral groups showed an improvement of simple motor behavior, such as apomorphine-induced rotation. However, only intranigral-grafted mice displayed significant improvements in fine motor skills evaluated in the staircase test and the trajectories of grasping-movement analysis. Until this study, contrasting results have been previously reported concerning functional recovery following intrastriatal or intranigral VM grafts. For instance, while some investigations have reported partial restoration in drug-induced rotation behavior in rodents after intranigral transplantation ,107,108,Intrastriatal-grafted dopamine neurons establish synaptic contacts with the host striatal neurons ,121, respars reticulata (SNpr) from the descending striato-nigral fibers. Specifically, it is now recognized that dendritic dopamine release plays a key role in the regulation of striato-nigral input (via D1-receptors), as well as in nigral output [The present study demonstrated that skilled forelimb movements were improved after a single L-DOPA injection in intrastriatal-grafted mice but not in lesioned mice. L-DOPA did not produce additional amelioration in intranigral-transplant mice, as this group spontaneously showed similar behavior to the control group. L-DOPA is the most effective and commonly used medication to alleviate motor symptoms for Parkinson’s disease. Previous studies in parkinsonian patients treated with L-DOPA have shown impressive efficacy, reducing tremor ,127,128 l output ,141,142.In light of these elements, it can be proposed that recovery of fine motor skills requires the near-complete restoration of the nigrostriatal circuitry and that intrastriatal transplantation or L-DOPA treatment alone do not lead to improvements in grasping movements. In contrast, intranigral transplantation may sustain the capacity to ameliorate complex motor behaviors without further need for L-DOPA therapy and its subsequent complications, such as tardive dyskinesia, possibly through dendritic dopaminergic control of SNpr activity and its ability to partially re-innervate not only the denervated striatum but also other nigral targets, such as the subthalamic nucleus or the globus pallidus, as we also reported here. This is of interest, as L-DOPA therapy is notorious for its decreasing beneficial effect with time, a drawback that might not be observed with intranigral transplantation, which restores physiological loss and fine motor skills, as shown here.Several mechanisms are responsible for the functional improvements observed following neural transplantation. Grafted cells could either directly integrate within the host brain or indirectly affect the host-injured microenvironment. Indeed, several studies, including our own, have demonstrated that grafted neurons integrate neuroanatomically into the host circuitries and promote the reconstruction of damaged connections ,143,144.In conclusion, by combining analyses of anatomical, electrophysiological, and both qualitative and quantitative aspects of fine motor skills, our results indicated that homotopic placement of immature dopaminergic neurons within the SNpc-lesioned site appeared to be an appropriate strategy to restore physiological functioning of the basal ganglia loops with a subsequent beneficial fine motor behavioral effect and no further need for L-DOPA adjuvant therapy. Whether this homotopic graft placement also restores nonmotor parkinsonian symptoms is yet to be determined.


The effects of freezing methods, including rapid freezing (RF) or slow freezing (SF), combined with thawing methods, e.g., water immersing thawing (WT) or cold thawing (CT), on the meat yield, drip loss, gel properties, and digestive properties of meat detached from red swamp crayfish were investigated. RF greatly reduced the freezing time compared to SF, and the thawing time of frozen crayfish was obviously shortened by WT in comparison to CT. RF and CT improved the meat yield but increased the drip loss, probably as a result of the greater protein denaturation or degradation. A soft and flexible gel was obtained by SF-CT, while a hard one was achieved by RF-WT. An SEM analysis showed that SF resulted in rough and irregular microstructures with larger pore sizes. Freeze-thawing led to an increase in the β-sheet content at the expense of α-helix and variations in the microenvironment of tyrosine and tryptophan residues in protein molecules of the gels, which was more pronounced in the SF-CT group. Moreover, freeze-thawing could cause enhanced protein digestibility but reduce the antioxidant activity of gels. These findings underline the promise of the freezing-thawing treatment in tuning the gel-based meat products of crayfish. Aquatic products are among the most important foods consumed worldwide due to their presence of high-value proteins, essential micronutrients, and desirable sensory properties (delicious taste and unique aroma) . HoweverThe ice crystals formed by freezing are disruptive to muscle fibers and cells of seafood, and they can induce protein denaturation, resulting in thawing drip loss and the deterioration of protein functional properties ,6. InterProcambarus clarkii) is an important economic species in China with high market value because of its nutritional attributes (high protein and low-fat content) and ease of breeding (fast growth rate and excellent adaptability) [The red swamp crayfish (ability) . In 2020ability) . The outability) . Nowadayability) .Meat separation is the essential stage for gel preparation. Its pretreatment method plays an important role in the subsequent deep processing of crayfish. Our preliminary experiments proved that freeze-thawing was effective for the shelling of crayfish. While freezing weakens the connected tissue, it will inevitably have some negative effects on the separated muscle, such as drip loss, freezing-induced protein denaturation, and decreased gelling properties . MoreoveTherefore, in this study, the effects of freezing methods, namely, liquid nitrogen rapid freezing (RF) and slow freezing (SF), combined with two thawing methods, including water-immersing thawing (WT) and cold thawing (CT), on meat yield and heat-induced gel properties of red swamp crayfish were investigated. The gel properties, including gel strength, deformation, and breaking force, color parameters, water-holding capacity, microstructure, protein secondary structural change, as well as the protein in vitro digestibility and antioxidant activity, were comprehensively evaluated to elaborate on the relationship between the gel properties and freezing-induced protein denaturation.P. clarkii) with weight of 15.93 ± 0.52 g were purchased in March 2021 from a local aquatic products market  and were immediately transported to the food processing laboratory within 1 h. The crayfish (n = 75) were kept alive at about 10 °C, and the subsequent experiments for crayfish were proceeded within 24 h.Live crayfish (n = 75) were randomly distributed into five clusters and were either (1) frozen by liquid nitrogen rapid freezing (RF), and thawed by water immersing thawing (WT) at about 10 °C, RF-WT; or (2) frozen by refrigerator slow freezing (SF) by using a BCD-216SDN refrigerator  at −18 °C, and thawed by WT, SF-WT; or (3) frozen by RF, and thawed by cold thawing (CT) in a BCD-216SDN refrigerator at 4 °C, RF-CT; or (4) frozen by SF and thawed by CT, SF-CT. The unprocessed crayfish were served as the control group, CG. The center temperature of crayfish treated by different freezing-thawing treatments was recorded by a multi-channel temperature logger  with a precision of ±0.2 °C.The live crayfish (Y) was defined as the mass ratio of obtained crayfish meat (W1) to crayfish (W0) and calculated as Equation (1).After the above freezing-thawing treatment, the crayfish muscle tissue was detached from the shells manually. The meat yield of crayfish .w/w) of NaCl was added to the paste and blended for 1 min at 1000 rpm for protein dissolution. The mixture was stuffed into beaker of 5 mL. Then the packed beakers were heated in a water bath , followed by cooling for 30 min. The crayfish meat gels were stored at 4 °C before further use.The peeled crayfish meat was chopped at 5000 rpm for 3 min by using a MQ5035 blender . Afterward, 2% , breaking force (g), and deformation (mm) of crayfish meat gel samples (about 15 mm in diameter and 15 mm in height.) were analyzed by using a TA. XT plus texture analyzer . The conditions were selected as follows: probe type, P/0.25S; test speed, 1.0 mm/s; pre-test speed, 1.0 mm/s; post-test speed, 5.0 mm/s; trigger force, 5 g; pressing distance, 12 mm. At least 6 samples were analyzed for each group. The color of crayfish meat gels was estimated by a colorimeter , and the test conditions were as follows: observer: CIE 10° standard observer; illuminant: Dv/v) at 4 °C for 24 h, then dehydrated with a series of ethanol solutions (60–100%). After substituting the ethanol with isoamyl acetate, the frozen gels were freeze-dried for 4.5 h in a model FD-ID-50 freeze dryer . The freeze-dried samples were sputter-coated, and the microstructure of crayfish meat gels was detected using a Carl Zeiss Microscope  at voltage of 3.0 kV.Water-holding capacity (WHC) of gels of crayfish meat was determined following the method of Liang, et al. . The craThe crayfish meat gels were diluted to 0.5 mg/L with 0.6 mol/L KCl solution, and the UV-Vis absorption spectrum at wavelength of 220–320 nm was recorded by D-7PC spectrophotometer . The second-derivative UV spectra was calculated and plotted by Origin 2018 software. For the FTIR analysis, the crayfish meat gels were frozen and then freeze-dried for 6 h by a freeze dryer . The freeze-dried meat gels were blended with potassium bromide of spectral purity, and the mixture was compressed into cylindrical sheet. The spectra of crayfish meat gels were measured in a Nicolet 6700 spectrometer . PeakFit software was applied to gain the protein secondary structures .In vitro gastrointestinal digestion of the crayfish meat gels was performed by the method of Singh, et al.  with somw/v, 36 mL) and homogenized  with a T18 homogenizer . The mixture was centrifuged  by a 5430R centrifuge , and the supernatant was separated and adjusted to a uniform protein concentration level. After digestion, 0.4 mL of the supernatant from the digested sample was used for analysis. Electrophoresis was performed by a DYCZ-24KF electrophoresis apparatus  at 170 V for about 3 h.SDS-PAGE was analyzed using the method of Li, et al.  with somp < 0.05. A minimum of 3 batches of samples were used for the test.All data were statistically analyzed by using the SPSS 20.0 software  by Duncan’s multiple range test at a significance level of The freezing curves of crayfish subjected to SF and RF are compared in Generally, the freezing curve of crayfish can be divided into three stages, i.e., pre-freezing stage , phase transition , and sub-freezing . Among those stages, stage II was considered the maximum-ice-crystal formation zone, which is critical for the size and distribution of ice crystals in frozen crayfish . As showAristichthys nobilis) fillets [Trachinotus ovatus) [The frozen crayfish were thawed by CT and WT until the core temperature reached 4 °C. The CT and WT curves of crayfish frozen by RF and SF are shown in  fillets , as well ovatus) . This mi ovatus) ,21. Like ovatus) .p < 0.05), indicating that the freeze-thaw process was capable of improving the meat yield. The muscle of crayfish is closely connected to the shell through connective tissue  [p < 0.05), implying that the protein denaturation in the RF-WT group was the lowest compared to the other treatments.As shown in mponent) . The fremponent) . Within p < 0.05). Evidently, crayfish frozen by SF exhibited higher drip loss than that of RF samples (p < 0.05). Thawing drip loss is related to the disruption of muscle fibers and cells by the mechanical stress of ice crystals (volume expansion) as well as the protein denaturation caused by pH changes during freezing [Changes in drip loss of crayfish samples treated by freeze-thawing are shown in freezing ,28. Therp < 0.05). As shown in The gel strength, deformation, and breaking force of crayfish meat gels as influenced by freeze-thawing treatments are shown in p < 0.05) than that of the SF (73.26 g•mm). For the CT, the gel strength of RF samples (57.30 g•mm) was also higher than that of SF (34.67 g•mm), though the difference is statistically insignificant (p > 0.05) (p < 0.05) than that of the CT by the same freezing method (RF or SF). In conclusion, RF and WT could preserve the mechanical properties of the crayfish meat gels compared to SF and CT.Moreover, it was found that crayfish meat gels with varied gel strengths could be obtained by different freezing and thawing combinations. In terms of thawing by WT, the gel strength of RF samples (244.56 g•mm) was significantly greater ( > 0.05) A. TherefCyprinus carpio) significantly decreased from 2.44 N to 1.94 N when subjected to a freeze-thawing process (frozen in a refrigerator at −25 °C and thawed in a refrigerator at 4 °C for 12 h). Subsequent trials found that the freeze-thawing caused MP denaturation and aggregation, as evidenced by the reduced protein solubility and Ca2+-ATPase activity, the increased particle size, and the high molecular weight of MP, as well as the increased average roughness of the MP film , leading to the reduced gel-forming ability. A previous study on the MP gelling properties of mirror carp treated with freeze-thawing (frozen at −25 °C and thawed at 4 °C for 12 h) found that freeze-thawing decreased the storage modulus G’ of MP gels, and the ζ-potential of MP declined from 26.87 mV to 23.9 mV, while the median diameter  of protein increased from 24.41 μm to 32.3 μm, implying protein aggregation of MP occurred during freeze-thawing [Li, Wang, Kong, Shi, and Xia  reported-thawing . ConsequThe freezing-induced denaturation of proteins could be ascribed to the ice crystal formation, freeze-concentration, and cold denaturation of proteins. Among those factors, protein cold denaturation was considered as the spontaneous unfolding of protein as a result of the exothermic process of decreased entropy during cooling, which could be almost negligible compared with the other two factors . Qian, HAlthough the mechanism of the impact of ice crystal formation on MP denaturation is currently not well disclosed, several clues are available for better understanding. During the process of freezing, the extracellular water begins to freeze, and the unfrozen water within the cells will migrate to the outside of the muscle fibers to form extracellular ice crystals. Then muscle fibers are severely compressed by the extracellular ice crystals, leading to the distortion of rigor bonds (actomyosin cross-bridges) and thereby the denaturation of myosin heads . This coIn fact, the thawing process is another crucial component affecting the gelling properties of frozen meat , becauseL, a, and b) of crayfish meat gels prepared from freeze-thawed crayfish were displayed in L values of the subsequent heat-induced gels (p < 0.05), compared with CG (untreated crayfish). Previous literature reported that the high value of lightness in gels could be related to protein aggregation, and the increased cross-links promote a compact structure, leading to a greater area of light reflectance [a and b values of crayfish meat gels (p < 0.05), making the gels appear attractive red, and this is consistent with the visual appearance as presented in The color parameters (lectance . The declectance . Howeverp < 0.05). Cando, et al. [C. carpio) treated with freeze-thaw cycles found that the immobilized water (T21 water), trapped in the microstructure of gels, was the major component in the gels, and its relaxation time became longer after freeze-thawing, suggesting that the amount of free and mobilized water in MP gels increased after a freeze-thawing treatment. This might be due to the loose and porous structure of MP gel because of the freezing-induced denatured proteins [The impact of the freeze-thawing process on the WHC of gels was shown in , et al.  reportedproteins .Portunus trituberculatus) significantly increased from 6.67 mg/100 g (fresh meat) to 8.12 mg/100 g and 7.53 mg/100 g, respectively, after air thawing (at 15 °C for 270 min) and water-immersing thawing (at 15 °C for 101 min). Interestingly, slow freezing rendered the gel network rough and irregular  MPs dissolve to form protein sol under the action of salt , comprising of myosin and actin separation and depolymerization of myosin chains; (ii) the dissociated MPs undergo thermal conformational changes and aggregations under conditions of heat. The stage ii is probably as follows: Myosin S1 expands when the temperature rises to around 35 °C, and myosin molecules begin to form dimers and oligomers through head-to-head agglutination, mostly through disulfide bonds and hydrophobic contacts. When the temperature rises to around 40 °C, the myosin dimers and oligomers tend to assemble due to head-to-head contacts. The tail radiates into three-dimensional space, whereas the aggregation head is prone to aggregating into spherical masses. As the temperature continues to rise above 55 °C, the formed aggregates are crosslinked and aggregated by tail interactions to form particle units, mainly via hydrogen bonds, by forming a three-dimensional network structure ,33,35.The formation of the microstructure was mainly influenced by the relative rates of protein aggregation and unfolding, i.e., a compact and homogenous gel microstructure is usually formed while protein unfolding proceeds slower than its aggregation. Conversely, a coarse and heterogeneous gel microstructure is commonly obtained when the rate of unfolding exceeds that of aggregation ,36. ThusThe UV spectra can be used to reflect the change of protein conformation by evaluating the microenvironment changes of aromatic amino acid side chains (such as tyrosine and tryptophan) . As show−1 , 2920 cm−1 , 1622 cm−1 , 1521 cm−1 , 1238 cm−1 , and 1071 cm−1 (C–O and C–C stretching) [−1 is often applied to estimate the information of protein secondary structures, including α-helix, β-turn, β-sheet, and random coil [The FT-IR spectra for crayfish meat gels as influenced by freeze-thawing treatments are displayed in etching) . The migetching) . Commonldom coil .p < 0.05) at the expense of α-helix, presumably due to the disruption of hydrogen bonding in α-helix by large ice crystals during slow freezing. It has been reported that freeze-thaw treatments could promote the conversion of α-helix (ordered rigid structure) to a relatively loose β sheet (disordered flexible structure) in the MP of mirror carp (Cyprinus carpio L.), owing to the broken hydrogen bonds by physical effects as well as the protein oxidation accelerated by freeze-thawing [The secondary structures of the meat gels of crayfish were estimated, and the results are displayed in -thawing . Therefop < 0.05). Meanwhile, the frozen crayfish thawed by CT showed a significant increase in protein digestibility of gels by comparison with WT (p < 0.05). The interference of SF and CT on digestion of crayfish meat gels could be mainly attributed to the denaturation or degradation of crayfish proteins, which altered the microstructure of the meat gels, increasing the accessibility of enzymes to hydrolytic sites [The effect of different freeze-thawing methods on the protein digestibility of crayfish meat gels by gastrointestinal environment simulation was shown in ic sites .p <0.05), particularly for the SF-CT  surimi gels induced by microbial transglutaminase (MTGase) retarded the pepsin digestion process at first, and the digestion sites and the quantities of peptides in the gels differed from the surimi gels without the addition of MTGase. However, other factors, such as molecular conformation, chemical bonds, etc., affecting the digestive characteristics (digestibility and produced peptides) of crayfish meat gels are still unclear.For the antioxidant activity of the digest from crayfish meat gels, it was found that freeze-thawing treatments significantly decreased the DPPH-RSA, ABTA-RSA, and FRAP of the gels compared with the CG group , and WT obviously decreased the thawing time compared with CT. However, SF and CT increased the meat yield, probably due to the greater degree of protein denaturation. Different combinations of freezing-thawing resulted in the crayfish meat gels with various mechanical properties. A soft and flexible texture could be obtained by SF-CT, while a hard and flexible gel could be produced by RF-WT. Freeze-thawing not only affects the internal microstructure, but also the tertiary and secondary structures of protein in the crayfish meat gels. Moreover, those changes might increase protein digestibility but reduce the antioxidant activity of gels. These findings could provide a scientific basis for the manufacture of a novel gel-based meat product made from crayfish. The mechanism of freeze-thawing treatment on the gelling properties of MP remains to be further studied.


The risk of a residual pad weight >10 g after the rehabilitative program significantly increased according to PV (p = 0.001) and the baseline pad weight (p = 0.002 and < 0.0001). In particular, PV > 40 cc and a baseline pad weight >400 g significantly  and independently predicted a 5.7 and a 35.4 times increase in the risk of a residual pad weight at the three-month follow-up, respectively. Conclusion: This is the first prospective trial whose primary objective is to verify the possible predictors, such as PV, that are able to condition the response to a pelvic floor rehabilitation program for UI after RP.Objectives: The objective of this study was to analyze the pre-operative and intra-operative variables that can condition urinary incontinence (UI) after radical prostatectomy (RP), as well as continence rate recovery during a pelvic floor rehabilitation program. Materials and Methods: A total of 72 cases with UI after RP were prospectively examined. All cases were homogeneously treated by the same surgeon, using the same RP technique. A combination of biofeedback (BF) and pelvic floor electric stimulation (PFES) performed by the same clinician and using the same protocol was used. Clinical, pathologic and surgical variables were analyzed in terms of 24 h pad test results (pad weight and pad-free status). Results: Prostate volume (PV) strongly varied from 24 to 127 cc (mean ± SD 46.39 ± 18.65 cc), and the baseline pad weight varied from 10 to 1500 cc (mean ± SD 354.29 ± 404.15 cc). PV strongly and positively correlated with the baseline pad weight (r = 0.4215;  Although there have been improvements in radical prostatectomy (RP) for prostate cancer (PC), this surgical procedure remains significantly associated with the development of urinary incontinence (UI). The rates of UI after RP significantly vary from 5 to 40% in different trials, depending on the characteristics of the populations and on the methods used ,2,3. UI aim of the present prospective trial was to analyze the pre-operative and intra-operative variables that can condition UI after catheter removal at RP and continence rate recovery during a pelvic floor rehabilitation program. In particular, in a population of PC cases with UI after RP, we prospectively evaluated whether pre-operative clinical characteristics, surgical techniques of RP and pathologic outcomes are able to significantly and independently influence baseline pad weight, pad weight improvement and pad-free status during the treatment with a combined program of BF and PFES and at 3-month follow-up.The This is a prospective trial on PC patients submitted to RP with post-operative UI. A real-life setting was analyzed at our Urological Department, using homogeneous criteria for the management of PC and UI.Patients with a histological diagnosis of prostatic adenocarcinoma considered for RP as a primary therapeutic option and presenting persistent UI at 30 days after catheter removal were consecutively included in the analysis. The protocol was approved by our internal ethical committee, and all patients gave their informed consensus for each procedure. All diagnostic and therapeutic procedures reflected our routine clinical practice in a department at high volume for the management of PC disease. Inclusion criteria were the following: histological diagnosis of adenocarcinoma, no distant metastases at clinical staging, RP chosen as primary treatment option, estimated life expectancy of ≥10 years, persistent UI (urinary leakage > 5 g at pad test) at 30 days after catheter removal and referred by the patient as able to influence his quality of life. Exclusion criteria were the following: prior bladder or prostate surgery, prior urinary of fecal incontinence, neurogenic dysfunctions, history of overactive bladder, neurological conditions, psychiatric therapies or other drugs able to influence bladder function, peri-operative complications, post-operative urinary stricture and early post-operative PC progression with the need for adjuvant treatments. No patient was prescribed anticholinergic drugs (or other drugs able to influence urinary continence) during the period of analysis. From January 2019 to January 2021, seventy-two consecutive cases corresponding to our inclusion and exclusion criteria were included in the study.The whole population of 72 cases is described in All cases were submitted to the same program of BF + PFES performed by the same clinician with more than 10 years of experience. In all cases, the program started 30 days after catheter removal and after the determination of baseline pad weight. The BF + PFES program has been described in previous articles ,24. PatiThirty days after catheter removal (baseline), during the treatment (BF + PFES) and at 3-month follow-up, urinary continence status was homogeneously assessed in terms of the 24 h pad test and pad weight (in grams). No pad use (pad-free status) or less than 2 g at pad test defined continence .p < 0.05.For statistical evaluation, the SPSS Statistics 1.6 program  was used. Descriptive statistical methods, such as number of cases, mean ± SD, median and range, were used. For the comparison of quantitative data and pairwise intergroup comparisons of variables, a Mann–Whitney test, Student’s t-test and one-way ANOVA test were performed. Pearson correlation analysis was also performed. Univariate and multivariate Cox proportional analyses to show the significant and independent role of the different variables in determining continence recovery were used. Primary outcomes were the following: baseline pad weight at catheter removal after RP, pad weight variation during pelvic floor program and pad-free status at 3-month follow-up. Statistical significance was evaluated at The baseline characteristics of our population are described in p = 0.0466) and inversely with pad-free status at follow-up . PV strongly and positively correlated with baseline pad weight  and inversely with 12-week pad weight  and pad-free status .According to Pearson analysis , only mep = 0.0009) varied between the two groups, with a median value of 174 cc in cases with PV ≤ 40 cc and of 360 cc in cases with PV > 40 cc. The pad weight during rehabilitation treatment and follow-up was always significantly (p < 0.01) lower in the group with PV ≤ 40 cc, with a higher percentage of reduction from baseline to 12-week follow-up  and, finally, a higher percentage of pad-free cases  (The whole population was stratified on the basis of PV into two groups: ≤40 cc (40.3% of cases) and >40 cc (59.7% of cases) . Accordi= 27.9%) a,b.p > 0.05) difference in terms of clinical and pathologic characteristics among the three groups was found, except for PV, which progressively and significantly (p = 0.0001) increased according to the baseline pad weight. A significantly (p = 0.0499) lower percentage of cases submitted to the NS RP technique showed an elevated baseline pad weight over 400 g (3.8%) when compared to cases with a baseline pad weight <100 g (28.6%). Pad weight during rehabilitation treatment and follow-up was always significantly (p < 0.0001) lower in the group with a baseline pad weight < 100 g, with a higher percentage of reduction from baseline to 12-week follow-up  and, finally, a higher percentage of pad-free cases  , 101–400 g (34.7% of cases) and >400 g (36.1% of cases) . No sign = 8.0%) b.p > 0.05) influence pad-free outcome after the rehabilitation program. Pad weight was always significantly (p < 0.001) lower at each follow-up interval in cases where pad-free status was obtained, and the percentage of pad weight reduction from baseline to 3-month follow-up was 99.8% (A higher percentage of cases with PV < 40 cc (55.2%) and a baseline pad weight < 100 g (76.2%) reached a pad-free status at the 3-month follow-up. No other clinical or pathologic variables were able to significantly (as 99.8% .p = 0.001) and the baseline pad weight (p = 0.002 and <0.0001). In particular, in the multivariate analysis, PV > 40 cc and a baseline pad weight > 400 g significantly  and independently predicted a 5.7 times and a 35.4 times increase in the risk of a residual pad weight > 10 g at the three-month follow-up, respectively.To the best of our knowledge, this is the first prospective clinical trial whose primary objective was to verify the possible predictors able to condition the response to a pelvic floor rehabilitation program for UI after RP. EAU guidelines underlined that there is some conflicting evidence on whether the addition of BF increases the effectiveness of PFME alone and whether PFES may add benefit in the short term . In a reThe strengths of this study are the following: (I) the present trial prospectively considered, as the primary objective, the analysis of predictors and variables that are able to condition results after a rehabilitative program for UI after RP; (II) all cases were homogeneously treated by the same surgeon with a high level of experience, using the same surgical technique for RP; (III) all cases underwent catheter removal after RP and started a PF rehabilitative program at the same time interval; (IV) all programs were homogeneously performed by the same clinician with a high level of experience, using the same protocol for BF + PFES; (V) the use of the 24 h pad test with pad weight determination, which is an objective and recommended test.However, some limitations deserve mention: (I) the number of cases could be higher and (II) the follow-up could be prolonged. However, most of the changes during these rehabilitative programs are obtained within the first 3 months.p < 0.01) higher percentage of cases with PV < 40 cc (55.2%) and a baseline pad weight < 100 g (76.2%) reaches pad-free status at the three-month follow-up. In our experience, most of the clinical, pathological and surgical variables are not able to significantly condition the results of a BF + PFES program for UI after RP. PV is the main variable significantly correlated with pad weight results, and an increased PV is able to significantly and independently increase the risk of higher baseline post-operative pad weights, as well as the risk of residual pad weight > 10 g after the rehabilitative program. A significantly (Future studies should always stratify results in terms of pre-operative variables, in particular, prostate volume and post-surgical pad weight, in order to better understand the results among the different non-invasive treatment strategies.


To conclude, four out of every five fully vaccinated individuals are estimated to be protected from contracting SARS CoV-2 infection. Vaccination lowered LOS and chances of development of severe disease.Effectiveness of corona virus disease-19 (COVID-19) vaccines used in India is unexplored and need to be substantiated. The present case-control study was planned to elicit the effectiveness of COVID-19 vaccines in preventing infection and disease severity in the general population of Bihar, India. This case-control study was conducted among people aged ≥45 years during April to June 2021. The cases were the COVID-19 patients admitted or visited All India Institute of Medical Sciences (AIIMS), Patna, Bihar, India, and were contacted directly. The controls were the individuals tested negative for severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) at the Virology laboratory, AIIMS-Patna and contacted telephonically for collection of relevant information. The vaccine effectiveness (VE) was calculated by using the formula (VE = 1 – odds ratio). The adjusted VE for partial and full vaccination were estimated to be 52.0%  39.0–63.0%) and 83.0% (95% CI 73.0–89.0%) respectively for preventing SARS CoV-2 infection. The sub-group analyses of the cases have shown that the length of hospital stays (LOS) (partially vaccinated: 9 days  Research in context:Evidence before this studyWe searched PubMed using the following search terms for observational studies (‘ChAdOx1 nCoV-19’ or ‘Oxford’ or ‘AstraZeneca’ or ‘BBV152’) and (‘COVID-19’ or ‘SARS-CoV-2’) and (‘vaccine’ or ‘vaccination’) and ‘effectiveness’ with no language or year restriction on 23 July 2021. We retrieved VIVALDI cohort study from the UK which reported that protection offered by a single-dose Oxford-AstraZeneca vaccine from SARS-CoV-2 infection to be 68% (at 35–48 days) among adults aged ≥65 years. Another cohort study from north-west London documented 74% risk reduction of testing positive for SARS-CoV-2 after 28 days of receiving first dose of ChAdOx1 nCoV-19. Public Health England test-negative case-control study observed vaccine effectiveness (VE) in terms of COVID-19-related symptom prevention of first dose of Oxford-AstraZeneca vaccine to be 60% between 28 and 34 days which increased to 73% after 35 days among older adults aged ≥70 years.Added value of this studyThrough adaptation of case-control design, we investigated the combined effectiveness of in use Covishield (Oxford-AstraZeneca) and Covaxin (Bharat Biotech) SARS-CoV-2 vaccines of India. Unadjusted combined VE was found to be 45.0% (95% CI 30.0–56.0) in the partially vaccinated group and 77.0% (95% CI 65.0–84.0%) in the fully vaccinated group in preventing SARS-CoV-2 infection. After adjusting with potential confounders, the VE for partial and full vaccination were estimated to be 52.0% (95% CI 39.0–63.0%) and 83.0% (95% CI 73.0–89.0%), respectively. We also adjusted VE with COVID-appropriate behaviour as per World Health Organisation (WHO) recommendations which other prior studies ignored. Vaccination was also found to confer protection against experiencing a severe disease and lowered hospital stay.Implications of all the available evidenceThis is one of the earlier evidences on real-world VE of COVID-19 vaccines used in the world's largest COVID-19 vaccination drive. Addition of the evidences generated in the present study with prior pieces of evidences available in this context will help in restoring faith regarding SARS-CoV-2 vaccination among the beneficiaries of it. This will also consolidate trust of policymakers on vaccination who issued emergency approval to these vaccines on public interest amid pandemic.Vaccination is one of the safest and cost-effective public health interventions for infectious disease prevention and control, especially in a pandemic situation . The corIndia started the world's largest vaccination drive with the use of the Covaxin and the Covishield vaccine both in a phased manner against COVID-19 on 16 January 2021 . In the Both the COVID-19 vaccines in India demonstrated good clinical efficacy and received the approval from the Drug Controller of India (DGCI) for emergency use. However, the real-world effectiveness of these vaccines have been largely unexplored and not documented in Indian settings till date , 8–10. MWe conducted an unmatched case-control study at All India Institute of Medical Sciences (AIIMS), situated in the ancient city of Patna beside the holy river Ganges between April and June 2021. All consecutive SARS CoV-2-positive patients who have availed services from our institute and the individuals who were tested negative for SARS CoV-2 at our Virology laboratory during the study period were the potential cases and controls for the study, respectively. We considered only the first visit of the cases for enrolment. Notably, none of our recruited cases were reinfected during the study period which was one of the pre-set exclusion criteria. In either group, only one participant from a family having multiple eligible participants (index-one) was considered. The controls with a history of COVID-19 or influenza-like illness (ILI) in the preceding 3 months from the day of data collection were excluded. The Institutional Ethical Committee (IEC) of AIIMS, Patna (Ref. AIIMS/Pat/IEC/2020/706) approved the study protocol.vs. control: 5.7% vs. 17.4%; power: 100.0%); partial vaccination ) [The odds ratio (OR) of contracting infection following COVID-19 vaccination was calculated to be 0.3 with the anticipated VE of 70%. During April 2021, the first dose COVID-19 vaccination coverage among the eligible general population of Bihar was found to be 3.7%. Considering the vaccination coverage (3.7%) and OR (0.3), the ratio of controls to cases 2:1, 95% precision and 80% power, the minimum sample size for the study was calculated to be 507 cases and 1013 controls using the online software OpenEpi , 19, 20.We approached consecutive COVID-19 patients aged ≥45 years (as during protocol development vaccination for 18–44 had not started) with a valid report (rapid antigen (Ag) or reverse transcriptase polymerase chain reaction (RT-PCR) for SARS-CoV-2) who have attained COVID dedicated flu clinic of our institute during the study period. Consented individuals were included as cases and were interviewed face-to-face. The information for severe and moribund cases was obtained from accompanying family members or friends directly. The highest disease severity of hospitalised cases was later retrieved from their medical records, while for home isolated ones, it was inquired telephonically. Line lists of RT-PCR-negative individuals aged ≥45 years were collected on a daily basis from our Virology laboratory during the study period. We telephonically approached individuals from these line lists at random within 3 days of their report and the consenting individuals were included as controls. All interviews were done by trained resident doctors of our institute. Details of case and control recruitment process are depicted in The study schedule was largely adopted from the World Health Organization (WHO) guidelines on the evaluation of COVID-19 effectiveness (2021) and modified in the local context . The finMild: No complaints of breathlessness and normal pulse oxygen saturation (SpO2): ≥94% and respiratory rate (RR): <24 breaths per minute (bpm); Moderate: Complaint of breathlessness and/or SpO2: 90–93% on room air and/or RR: 24–30 bpm with no features of severe disease ; Severe: SpO2: <90% on room air and/or RR: >30 bpm and/or features of severe disease  [unvaccinated: At the time of testing for SARS-CoV-2, one has not received jab or was within 14 days of first jab; partially vaccinated: did the testing for SARS-CoV-2, ≥14 days of first jab or before 14 days of second jab; fully vaccinated: did the testing for SARS-CoV-2, ≥14 days after second jab.Severity of COVID-19, unction) , 23. FouU test and Kruskal–Wallis test were used to compare medians across binomial and ordinal variables, respectively. Bonferroni's test was used for post-hoc analysis of ordinal variables. Pearson's χ2 test was used to test the association between categorical variables. The χ2 adjusted standardised test was used for higher order contingency table (>2 × 2) to identify significant pair. The protective effect of COVID vaccines for contracting COVID-19 infection was statistically tested using univariable logistic regression followed by multivariable logistic regression, and quantification was expressed as OR. We adjusted for all actual or potential confounders  in the multivariable logistic regression model. Unadjusted and adjusted VE were calculated using the following formulae: (VE = (1 – OR) × 100%). We used 95% confidence level for all the statistical tests used.The information collected was entered using Epicollect5 (an open-source data collection software) . The datThe median age of the cases was higher than the controls while male gender constituted about two-third of the study population in both the groups. About three-fifth (58.8%) of the cases and one-fourth of the controls (28.8%) were having at least one chronic co-morbidity. About two-fifth of the cases (42.1%) and three-fifth of the controls (61.4%) have received at least a single jab. About two-third and one-fifth of the study participants in both the groups have received Covishield and Covaxin, respectively .Table 1Inaccessibility to vaccination centres  was reported to be the predominant reason for not getting vaccinated followed by fear of side effects  and sceptical about VE  .Fig. 2.Unadjusted VE was found to be 45.0% (95% CI 30.0–56.0) in the partially vaccinated group and 77.0% (95% CI 65.0%–84.0%) in the fully vaccinated group in preventing SARS-CoV-2 infection. After adjusting with potential confounders like age, sex, occupation, COVID-inappropriate behaviour score, chronic co-morbidity, H/O hospitalisation for chronic co-morbidity, ILI, past COVID-19 infection and high-risk contact with a COVID-19 case or suspect, the VE for partial and full vaccination were estimated to be 52.0% (95% CI 39.0–63.0%) and 83.0% (95% CI 73.0–89.0%), respectively. A full vaccination course was calculated to have provided 31.0% additional protection compared to partial vaccination in preventing SARS-CoV-2 infection .Table 2P = 0.028). The fully vaccinated COVID patients had a lower probability of having the severe disease on arrival (18.2%) compared to the partially vaccinated (31.8%) and the unvaccinated (42.6%) cases (P = 0.022). Overall, the fully vaccinated COVID patients were also found to have less likelihood of experiencing severe disease (30.3%) during their illness compared to partially vaccinated (51.3%) and unvaccinated (54.1%) cases (P = 0.035). The final outcome (death/recovery) was statistically indifferent across the different vaccination groups ) and unvaccinated (12 days (IQR 6–16 days)) was statistically significant . We have also adjusted for the confounders in the final analysis. Most importantly, we have adjusted for the COVID-19-appropriate behaviour in our study.Like any other observational study, our study also has few limitations. First, we recruited cases only from one centre, AIIMS, Patna which is the highest referral institute for the management of COVID-19 in Bihar. Patients from all the corners of the state are being referred here. Thus, the cases of the present study might represent COVID-19 cases for the state of Bihar to a large extent. Second, the telephonic interview was used as a mode of data collection for home-isolated cases and controls. Inherent biases associated with the telephonic interview might be present. The telephonic interviews were made by trained resident doctors, and repeat calls following consent were made in case of any doubt over a piece of information. This might have alleviated the bias to some extent. Third, the errors associated with self-reporting of some information. We tried to reduce these errors by developing rapport with the participant and allowing sufficient time to respond. We also requested them to confirm the vaccination details by cross-checking with the CoWIN app or the message received following vaccination. Fourth, the misclassification due to the inherent properties of the test used for the investigation of SARS-CoV-2. The RT-PCR-based testing is only 60–70% sensitive, although specificity is claimed to be more than 90%. Lastly, we did not have a sufficient sample size to do sub-group analysis for Covishield and Covaxin groups. However, the analysis and findings with the samples at our hand are provided in Supplementary Tables.COVID-19 vaccination was found to be effective in infection prevention. One out of two and four out of five individuals were found to be protected against SARS-CoV-2 infection following partial and full vaccination, respectively. The vaccinated individuals had lesser LOS compared to unvaccinated ones. Additionally, the fully vaccinated individuals were less likely to develop severe disease. We recommend studies with a larger sample size to elicit the VE of individual COVID-19 vaccines.